The regulation of the cell number in multicellular organisms is an essential requirement both for the development of the organism and the maintenance of its different vital functions. Apoptosis is a process in which cells that are not required at a certain stage of development undergo a programmed cell death. For example, it is the precisely regulated interplay of proliferation and apoptosis that is responsible for shaping tissues and organs in the developing embryo. Apoptosis is induced by a number of different stimuli and propagated by different pathways.
The extrinsic pathway inter alia includes binding of the death receptor ligands, e.g. TNF, TRAIL and CD95L, to their respective receptor molecules in the cell, which binding ultimately activates caspase-8. The intrinsic pathway is stimulated, for example, through application of the DNA-damaging topoisomerase inhibitors doxorubicin, etoposide and staurosporine, TCR stimulation, irradiation, DNA damage and the like, which results in the release of cytochrom c from mitochondria, activation of caspase-9, and the granzyme B-dependent route to caspase activation. Granzyme B can process BID as well as caspase-3 and -7 to initiate apoptosis. These pathways are linked so that they affect each other. Cells which undergo apoptosis regularly show a number of characterizing features, such as chromatin condensation, cell shrinkage, increased membrane permeability and internucleosomal DNA cleavage.
Apart from its different functions during development, an impaired, increased or defective apoptosis is also involved in a number of diseases or pathological conditions. For example, ischemia and subsequent reperfusion induces apoptosis both via the intrinsic and extrinsic pathway which can lead to a complete failure o crucial tissues or organs the function of which are essential for the organism. For example, an abnormally increased extent of apoptosis may result in renal failure and brain tissue damage. Further, it has been reported that an increased apoptosis is also involved in gastrointestinal disorders. In contrast, an abnormally decreased apoptosis is involved in different forms of cancer.
Although different inhibitors of apoptosis have been suggested in the prior art, none of these approaches has proven suitable for clinical practice. For example, different anti-apoptotic proteins have been discussed as therapeutic compounds. However, the clinical use of these proteins is presently only possible using gene transfer methods which involve unforeseeable risks for the patients undergoing such treatment. Therefore, it is an objective of the present invention to provide means and methods for a save and efficient treatment of an abnormally increased apoptosis. This objective is achieved by a fusion protein comprising a protein transduction domain capable of introducing the fusion protein into a mammalian cell and an anti-apoptotic protein as further defined herein.
According to the present invention, it has now been surprisingly found that the coupling of the cowpox virus cytokine response modifier A (CrmA) protein to a protein transduction domain, such as the protein transduction domain of the HIV Tat protein, results in an anti-apoptotically active fusion protein which is effective in blocking apoptosis in a patient in need of an anti-apoptotic treatment, i.e. a patient suffering from a disease characterized by a pathogenic level of apoptosis in cells or tissues of the body.
Accordingly, a fusion protein is provided, said fusion protein comprising at least the following components:                (a) a protein transduction domain capable of introducing the fusion protein into a mammalian cell;        (b) an anti-apoptotic protein comprising the amino acid sequence of SEQ ID NO:1 or an anti-apoptotically active variant or fragment thereof.        
The use of therapeutically active proteins or polypeptides was for a long time hampered by the inability of these proteins or polypeptides to cross the cell membrane barrier. The discovery of protein transduction domains (PTDs) like that from the HIT Tat protein which act as shuttle molecules facilitated the transduction of large proteins or polypeptides into the target cell. Protein transduction domains enable the PTD-protein complex to enter a cell in an receptor-independent manner. Several different PTDs have been identified so far, for example in the Antennapedia protein of Drosophila, the VP22 protein of Herpes simplex virus and the TAT protein from HIV type 1. These PTDs generally consist of about 10-50 amino acids and share several structural similarities. One striking feature of PTDs is the possession of several basic amino acids which is indicative for a general mechanism by which these molecules enter the cell. It has been speculated that an interaction of heparan structures on the surface of the target cell with the basic amino acids in the PTD sequence result finally in the transduction of the whole molecule into the cell.
As used in the context of the present invention, the term “fusion protein” refers to a non-naturally occurring hybrid protein comprising at least two different proteins or protein fragments. The fusion protein of the present invention comprises a protein transduction domain which is suitable for introducing the fusion protein into a cell, preferably into a mammalian cell of a patient which is in need of an anti-apoptotic treatment. Preferably, the patient is a human patient and the cells into which the fusion protein of the invention shall enter via the PTD is a human cell, for example, a cell of an organ tissue, such as renal tissue. In principle, any PTD may be used which does not adversely affect folding of the anti-apoptotic protein to which it is fused or otherwise coupled. This means, the PTD should not interfere with the anti-apoptotic activity of the protein comprising the amino acid sequence of SEQ ID NO:1 or an anti-apoptotically active fragment or variant thereof to an extent that the desired anti-apoptotic activity is abolished. It is understood, however, that a loss of activity of the anti-apoptotic protein of 20%, 30%, 40%, 50%, 60%, 70, or even up to 80% may be tolerated in cases where a particular fusion protein exhibits other favorable characteristics, such as an increased stability or shelf-life or improved. pharmacological properties, such as a decreased immunogenicity. The skilled person will be readily able to assess whether a given fusion protein is suitable for being used in a desired therapeutic context.
According to a preferred aspect of the invention, the protein transduction domain used in the fusion protein is derived either from the Antennapedia protein of Drosophila, the VP22 protein of Herpes simplex virus or the TAT protein from human immunodeficiency virus (HIV) type 1. According to a particularly preferred embodiment, the fusion protein comprises a protein transduction domain which is derived from the transduction domain of the Tat protein of HIV type 1. The transduction domain of the Tat protein of human immunodeficiency virus type 1 has been described in the art in several publications, for example, in Bhorade, R. et al. (2000), Bioconjug. Chem. 11:301-305; Becker-Hapak, M. et al. (2001), Methods 24:247-256; and Dietz, G. et al. (2002), Mol. Cell Neurosci. 21:29-37. Mutagenesis studies showed that a core structure of 11 amino acids of the HIV Tat transduction domain is sufficient for the efficient transfer of proteins across the cell membrane. The minimal structure of the HIV Tat transduction domain is represented by the 11-amino acid sequence YGRKKRRQRRR (SEQ ID NO:3). Thus, in a preferred embodiment, the fusion protein comprises a protein transduction domain which comprises or consists of the amino acid sequence of SEQ ID NO:3 or an amino acid sequence having at least 80%, preferably at least 95%, identity thereto.
The fusion protein of the invention also comprises, as a further component, the amino acid sequence of SEQ ID NO:1 or an anti-apoptotically active fragment or variant thereof. The amino acid sequence set forth in SEQ ID NO:1 describes the primary structure of the cytokine response modifier A (CrmA) protein from the cowpox virus. This protein has been shown to be an efficient suppressor of apoptosis. By developing an anti-apoptotic protein during evolution, the cowpox virus has gained the ability to protect the host cells which are used by the virus for propagation. Unlike other apoptosis suppressors, the effect of CrmA is based on the simultaneous inhibition of caspases-1 and -8 of the extrinsic pathway, caspase-9 of the intrinsic pathway, and executor caspases-3, -6 and -7. Due to this manfold mode of action, CrmA is a particularly powerful inhibitor of apoptosis.
Several attempts have been made in the art to use the CrmA protein for blocking apoptosis both in in vitro systems and in animal models. These attempts were based on the heterologous expression of the CrmA protein in cell lines or transgenic animals, which has the fundamental disadvantage that CrmA expression cannot be shut off once the expression construct has been introduced into the target cell. The particular advantage of the present invention resides in the fact that the inhibition of an undesired apoptosis is feasible for a limited period of time. The fusion protein of the invention allows for the temporary inhibition of apoptosis in situations where transient suppression of cell death is desired. It has been shown that the fusion proteins of the invention exhibit a half life time of approximately 8 hours, so that the extent and duration of treatment can be readily adjusted by the frequency of administration of the fusion protein. In other words, the introduction of the CrmA protein by use of a protein transduction domain enables the practitioner to adjust the anti-apoptotic treatment to the clinical situation of the patient to be treated.
As will be appreciated by the person of skill, the cowpox virus is subject to permanent modification due to the high mutation rate of the viral genome. As a consequence, several amino acid sequences are available in the databases for the CrmA protein which differ one or more amino acids from the amino acid sequence in SEQ ID NO:1. These variants of CrmA are comprised by the present invention provided that they have an anti-apoptotic activity. As used herein, variants of the protein of SEQ ID NO:1 are proteins with an amino acid sequence that differs from the amino acid sequence of the protein of SEQ ID NO:1 by one or more amino acid substitutions, additions or deletions. In general, any amino acid residue of the sequence shown in SEQ ID NO:1 may be substituted by another amino acid, provided that the resulting protein variant still exhibits an anti-apoptotic activity. Determination of the anti-apoptotic activity can be performed, for example, by the method described in Wender et al. (2000),Proc. Natl. Acad. Sci USA 97 (24):13003-8. In particular, protein variants are included which differ from the amino acid sequence shown in SEQ ID NO:1 in up to 5, 10, 15, 20 or even up to 25 amino acid positions.
It is preferred that the substitutions are conservative substitutions, i.e. substitutions of an amino acid residue by another amino acid of a similar polarity, which acts as a functional equivalent. Preferably, the amino acid residue used as a substitute is selected from the same group of amino acids as the amino acid residue to be substituted. For example, a hydrophobic residue can be substituted with another hydrophobic residue, or a polar residue can be substituted with another polar residue having the same charge. Functionally homologous amino acids which may be used for a conservative substitution comprise, for example, non-polar amino acids such as glycine, valine, alanine, isoleucine, leucine, methionine, proline, phenylalanine, and tryptophan. Examples of uncharged polar amino acids comprise serine, threonine, glutamine, asparagine, tyrosine and cysteine. Examples of charged polar (basic) amino acids comprise histidine, arginine and lysine. Examples of charged polar (acidic) amino acids comprise aspartic acid and glutamic acid.
Also comprised by the term “variant” are protein sequences which include more amino acids than the sequence of SEQ ID NO:1, i.e. protein sequences in which one or more amino acids have been inserted. Such insertions may in principle occur at any position of the protein of SEQ ID NO:1. The insertions may be stretches of contiguous amino acids comprising, for example, 2, 3, 4, 5, 6, 7, 8, 9 or ten amino acids. Similarly, the term “variant” also includes protein sequences in which one or more amino acids are deleted as compared to the protein of SEQ ID NO:1. The deletion can involve several contiguous amino acid residues of the protein of SEQ ID NO:1, for example, 2, 3, 4, 5, 6, 7, 8, 9 or ten amino acids. A particular preferred variant of the CrmA protein has the amino acid sequence shown in SEQ ID NO:2, This variant inter alia differs from the CrmA protein of SEQ ID NO:1 by lack of the valine-serine-alanine stretch shown in positions 55-57 of SEQ ID NO:1. This deletion does not affect the anti-apoptotic activity of the variant. Variants of the protein of SEQ ID NO:1, also include structural modifications, for example modified amino acids, such as amino acids that have been altered by phosphorylation, glycosylation, acetylation, thiolation, branching and/or cyclization.
The variants of the CrmA protein of SEQ ID NO:1, which have been modified by substitution, addition or deletion of amino acids, normally show a considerable degree of amino acid sequence homology or identity to the CrmA protein of SEQ ID NO:1. Preferably, the homology or identity on the amino acid level is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% if the sequence of the variant is optimally aligned with that of SEQ ID NO:1. Methods and computer programs for determining amino acid homology are well known in the art.
The present invention also contemplates the use of anti-apoptotically active fragments, which are either derived from the protein of SEQ ID NO:1 or its variants. Anti-apoptotically active fragments of SEQ ID NO:1 or its variants include peptides or polypeptides that differ from the amino acid sequence shown in SEQ ID NO:1 or from its variants by the absence of one or more amino acids at the N-terminus and/or C-terminus of the protein. For example, the CrmA component of the fusion protein used in the below examples lacks the methionin residue in position 1 of the sequence depicted in SEQ ID NO:1, which means that it actually represents a fragment of the sequence in SEQ ID NO:1. The person of ordinary skill will be able to determine further active fragments of the sequence depicted in SEQ ID NO:1 or its variants by performing routine methods described in great detail in the prior art.
Preferably, the fusion protein of the invention further comprises a linker between the protein transduction domain and the anti-apoptotic protein. The linker may comprise any number of amino acids as long as the anti-apoptotic activity of the CrmA portion is maintained and the introduction of the fusion protein into the cell via the PTD is still effected. The linker normally comprises 5-30 amino acids, preferably 8-25 amino acids, and more preferably 10-20 amino acids. The linker regularly includes several glycine residues, since this amino acid is less likely to affect protein folding of the active portion of the fusion protein. The fusion protein may also comprise additional components, for example, an affinity tag which facilitates binding of the fusion protein via a compound exhibiting binding affinity to the tag. For example, the affinity tag may be a poly-histidine tag comprising 6-12 histidine residues which specifically interacts with a nickel ion chelate matrix. The fusion protein which was used in the examples of the present invention (designated TAT-CrmA) and which is depicted in SEQ ID NO:4 comprises a tag of 6 histidine residues for purification. Alternatively, the tag may be glutathione-S-transferase allowing the purification on a glutathione matrix. Further affinity tags are well-known in the art. Non-liming examples for pairs of affinity tag and affinity ligand include maltose-binding-protein (MBP) and maltose; avidin and biotin; Streptag and streptavidin or neutravidin. Affinity tags may be attached to a molecule by fusion/ligation of cDNAs of the molecule of interest with the sequence encoding the tag. Where the affinity tag is a peptide or polypeptide, such tag may conveniently be expressed together with the fusion protein of the invention as a single expression product. In other embodiments, the affinity tag may be attached by chemical coupling reactions. For example, biotin may be chemically coupled to the fusion protein.
The fusion protein of the invention has been shown to be effective in inhibiting or blocking apoptosis when administered in the form suitable for the delivery of therapeutic proteins. Accordingly, the fusion protein provided by the invention is for blocking apoptosis in a patient in need thereof. The fusion protein may be used, for example, for treating diseases which involve a dysregulated, excessive level of apoptosis, such as reperfusion injury, stroke, myocardial infarction, toxic and ischemic organ failure, fulminant hepatic failure, AIDS, neurodegenerative diseases, graft versus host disease, alcoholinduced liver cirrhosis, spinal cord injury, acute leukemia, or viral hepatitis.
Preferably, the fusion protein of the present invention is administered as a pharmaceutical composition. Accordingly, the invention also provides a pharmaceutical composition comprising a fusion protein as defined above. The pharmaceutical composition is for blocking apoptosis in a patient in need thereof. In particular, the composition is for treating reperfusion injury, stroke, myocardial infarction, toxic and ischemic organ failure, fulminant hepatic failure, AIDS, neurodegenerative diseases, graft versus host disease, alcohol induced liver cirrhosis, spinal cord injury, acute leukemia or viral hepatitis. The preparation of pharmaceutical compositions comprising one or more therapeutic proteins is well known by those working in the field of pharmaceutics. Typically, such compositions are prepared for injection either as liquid solutions or suspensions. The active ingredient (i.e. the fusion protein) can be mixed with excipients which are pharmaceutically acceptable and compatible with the fusion protein when used in human patients. The pharmaceutical compositions of the present invention normally comprise a physiologically acceptable carrier together with the fusion protein dissolved or dispersed therein as an active ingredient. Pharmaceutically acceptable carriers comprise, for example, water, saline, Ringer's Solutions, or dextrose solution. Further suitable carriers for compositions comprising the anti-apoptotic fusion protein of the invention are described in standard textbooks, for example, in “Remington's Pharmaceutical Sciences”, Mack Pub. Co., New Jersey (1991). In addition to a carrier, the pharmaceutical composition of the invention may also comprise wetting agents, buffering agents, stabilizers, dyes, preservatives and the like in any concentration, provided that these compounds do not interfere with the anti-apoptotic activity of the fusion protein of the invention.
Different routes of administration are feasible for providing the fusion protein to the site or organ which requires the inhibition of apoptosis. Preferably, the pharmaceutical composition is formulated for systemic administration, for example, for parenteral administration. Parenteral administration may include intravenous, intradermal, intraarterial, subcutaneous, topical, transmucosal, or rectal administration. According to a particular preferred embodiment, the pharmaceutical composition is formulated for injection.
Pharmaceutical compositions suitable for injection normally include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The composition should furthermore be stable under regular conditions of manufacturing and storage. The composition intended for injection must be sterile in order to avoid a severe immunologic reaction in the patient receiving the injection. For maintaining sterility, the pharmaceutical composition normally includes preservatives, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like, to suppress microbial growth in the product. For intravenous or intraarterial administration, suitable carriers may comprise physiological saline, bacterio-static water, Cremophor EL™ (BASF) or phosphate buffered saline (PBS). The carrier may also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. A prolonged absorption of the injectable compositions can be achieved by including into the composition an agent which delays absorption, for example, aluminum monostearate or gelain. Sterile injectable solutions can be prepared by incorporating the active ingredient (the fusion protein) in the required amount in an appropriate solvent with one or a combination of the above mentioned ingredients followed by sterile filtration. Similarly, dispersions are prepared by incorporating the fusion protein into a sterile vehicle that contains a dispersion medium and optionally other ingredients as outlined above. Sterile solutions can also be obtained by providing the fusion protein in the form of a sterile powder by methods known in the art, such as vacuum drying or freeze-drying, and reconstituting the powder with a sterile liquid to yield the final solution for injection. Alternatively, the pharmaceutical composition according to the invention may also be administered by continuous infusion.
Administration of the pharmaceutical composition may also be achieved by transmucosal or transdermal delivery. For transmucosal or transdermal administration, the pharmaceutical composition comprising the fusion protein of the invention will comprise penetrants which are appropriate for crossing the skin or mucosal barrier. Such penetrants are known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art. Preferably, the compounds are prepared in the form of suppositories, with conventional suppository bases such as cocoa butter and other glycerides for rectal delivery.
In one embodiment, the active compounds are prepared with carriers that will protect the peptides against elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparing controlled release formulation are well-known in the art. Furthermore, sustained-release compositions may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers, which matrices are in the form of shaped articles, e.g., films or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels, polylactides, copolymers of L-glutamic acid and ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers and the like.
In a further aspect, the present invention provides a method of treating a pathological apoptosis in a patient in need thereof comprising the administration of a therapeutically effective amount of an anti-apoptotic fusion protein as defined above. Preferably, the patient is suffering from a disease which involves a dysregulated, excessive level of apoptosis, such as reperfusion injury, stroke, myocardial infarction, toxic and ischemic organ failure, fulminant hepatic failure, AIDS, neurodegenerative diseases, graft versus host disease, alcohol-induced liver cirrhosis, spinal cord injury, acute leukemia, or viral hepatitis.
A therapeutically effective amount of the anti-apoptotic fusion protein is typically an amount which—upon administration—is sufficient to effectively block or inhibit apoptosis in the target organ or tissue of the patient to be treated. Typically, the dosage of the fusion protein per body weight varies from about 0.1 mg per kg body weight of the patient to about 50 mg per kg body weight of the patient, and more preferably from about 0.5 mg per kg body weight of the patient to about 20 mg per kg body weight of the patient, and even more preferably from 1 mg per kg body weight of the patient to about 10 mg per kg body weight of the patient. The administration regimen may include one or more administrations of the protein daily. It will be appreciated by those skilled in the art that the concrete amount of the fusion protein which is to be administered to the patient will depend on several factors, such as age and weight of the patient, as well as from the nature and severity of the medical symptoms to be treated. The amount which exerts the desired therapeutic effect may be determined in each individual case by use of routine experimentation.
In a further aspect, the invention relates to a polynucleotide encoding a fusion protein as defined above. Polynucleotides which encode the fusion proteins of the invention may consist of DNA or RNA. The polynucleotide encoding the fusion protein of the invention refers to a DNA or RNA molecule which directs the expression of the desired fusion protein. An exemplary polynucleotide which codes for a fusion protein of the invention is depicted in SEQ ID NO:5; this polynucleotide encodes the TAT-CrmA fusion protein of SEQ ID NO:4. However, it is to be understood that other polynucleotides encoding the protein of SEQ ID NO:1, which differ from the sequence of SEQ ID NO:5 in one or more of the nucleotide position due to the degeneracy of the genetic code, are also included. Similarly, polynucleotides encoding variants or fragments of the protein of SEQ ID NO:1 are also included.
The polynucleotides encoding the fusion proteins of the invention may be cloned in an expression vector in order to provide means for recombinant preparation the fusion protein. Generally, expression vectors are self-replicating DNA or RNA constructs into which a polynucleotide of interest is inserted such that the coding sequence of the polynucleotide is operably linked to suitable regulatory elements which allow for the controlled expression of the coding sequence in a host cell. The specific control elements which are required for expression of the polynucleotide will depend on the host cell that is used and may include a transcriptional promoter, an operator, an enhancer to increase the mRNA expression level, a ribosome binding site, and suitable transcription and translation terminators. The promoter can be a constitutive or inducible promoter, for example, the commonly used promoters of galactokinase, uridylyltransferase, phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase, alcohol dehydrogenase, or viral promoters, such as the promoters from SV40, cytomegalovirus (CMV) or Molony murine leukemia virus (MMLV), and the like. Expression vectors may also contain an origin of replication for the autonomous replication of the vector within a host cell, and one or more selection marker which allow monitoring the transfection into the host cell.
The expression vector may be designed for expressing the recombinant protein in a prokaryotic or eukaryotic host. Preferably, the expression vector will nave one capability to stably replicate within the host cell so that the number of the polynucleotide which encodes the fusion protein of interest is increased within the cell. It as, however, also possible to use an expression vector which is unable to replicate within the cells and allows only transient expression of the fusion protein. Alternatively, expression vectors may be used which integrate into the genome of the host cell that is transduced or transfected with the expression vector. Various expression vectors are known in the prior art which are suitable for mediating expression of the anti-apoptotic fusion protein of the invention both in prokaryotic or eukaryotic host cells. Methods for introducing the expression vector into a host cell have also been extensively discussed in the literature. A variety of different methods may be used for transducing or transfecting the expression vector of the invention into a host cell, for example, electroporation, microinjection, transformation, transfection, protoplast fusion, microprojectile bombardment and the like.
Accordingly, the invention also relates to a host cell which comprises an expression vector harboring a polynucleotide encoding the fusion protein of the invention. The cells may be derived from prokaryotes, or from lower or higher eukaryotes. Suitable prokaryotes which can be transduced or transfected with an expression vector encoding the fusion protein of the invention include, for example, bacteria like Bacillus subtilis, Escherichia coli, and the like. Lower eukaryotes that may be used in the methods of the present invention include yeasts, in particular yeasts of the genus Saccharomyces, such as S. cerevisiae, S. bailii, S. bayanus, S. boulardii, S. carlsbergensis, of the genus Pichia, such as P. pastoris, P. methanolica, P. stipitis, or of the genus Dictyostelium, such as D. discoideum. Higher eukaryotes include animal cell lines, in particular mammalian cell lines, for example cell lines derived from rodents, primates and human. Useful cell lines for use according to the invention include Chinese hamster ovary (CHO) cell lines, baby rat kidney (BRK) cell lines, Madin Darby canine kidney (MDCK) cell lines, NSO myeloma cells, monkey kidney COS cells, human embryonic kidney 293 cells, and cancer cell lines such as SKBR3 cells, Jurket T cells or HeLa cells.
The host cells of the invention which comprise a polynucleotide encoding the anti-apoptotic fusion protein of the invention, preferably in the form of an expression vector suitable of directing expression in the respective host cell, may be conveniently used for preparing the fusion protein. Preferably, the expression vector is introduced into the host cell such that the expression of the fusion protein is effected by propagation of the host cells. Accordingly, the invention also provides a method for preparing a fusion protein as defined herein, comprising the culturing of a host cell which comprises the polynucleotide encoding the anti-apoptotic fusion protein or the expression vector including such polynucleotide under conditions which allow for the expression of the fusion protein.
The provision of the fusion proteins of the invention allows for the preparation of antibodies which hand to said fusion protein. The method comprises the immunization of a non-human animal with the fusion protein, and obtaining the antibodies produced in response to the fusion protein from the serum of said animal.
The invention therefore also relates to the use of a fusion protein as defined herein or a polynucleotide encoding such fusion protein for the preparation of a pharmaceutical composition for blocking apoptosis in a patient in need thereof. Similarly, the invention also relates to the use of an expression vector comprising a polynucleotide encoding a fusion protein of the invention and to a host cell comprising such a polynucleotide or expression vector for the preparation of a pharmaceutical composition for blocking apoptosis in a patient in need thereof. Preferably, these materials are for the preparation of a pharmaceutical composition for the treatment of reperfusion injury, stroke, myocardial infarction, toxic and ischemic organ failure, fulminant hepatic failure, AIDS, neurodegenerative diseases, graft versus host disease, alcohol induced liver cirrhosis, spinal cord injury, acute leukemia, or viral hepatitis.
Finally, the invention also contemplates a kit comprising the fusion protein of the invention, a polynucleotide encoding such fusion protein, an expression vector comprising a polynucleotide encoding such fusion protein and/or a host cell comprising such a polynucleotide or expression vector. The kit may comprise further materials which are useful for preparing a composition for administration to the patient. For example, the kit may include buffers, stabilizers, carriers and the like for mixing with the fusion protein prior to administration. The kit will further comprise instructions for using the components in the preparation of a pharmaceutical composition for blocking apoptosis in a patient suffering from an increased or dysregulated level of apoptosis. For example, the instructions will relate to the administration of the fusion protein to a patient, for example, in preferred dosage regimens. If the kit comprises an expression vector harboring a polynucleotide coding for the fusion protein of the invention, the instructions will further relate to the introduction of said vector into a suitable host cell and to conditions suitable for expressing and recovering the fusion protein of the invention.